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Hi Meg I would highly recommend giving 6M guanidinium chloride a go in parallel, to compare the solubilization efficiency of the two approaches. We have had a couple of cases in the lab where homologous proteins showed strong preference either to urea OR guanidinium chloride when it came to solubilization of their inclusion bodies for refolding protocols. best wishes Savvas ---- Savvas Savvides http://www.lprobe.ugent.be/xray.html -----Original Message----- From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of Joerg Standfuss Sent: Thursday, December 03, 2009 8:57 AM To: [log in to unmask] Subject: Re: [ccp4bb] Solubilization buffer Dear Meg, There is a database of refolding conditions from which you could get some inspiration. http://refold.med.monash.edu.au/search.php The Urea conditions I have seen there mostly use 37C. for solubilization/refolding and nearly all contain a reducing agent. Have a look. There are also a great number of other additives people use to improve things. Joerg > Hi all, > > We use 8M urea solubilization buffer for our protein in inclusion bodies > and > recommended temperature is 10-15º C. but in 8M conc the urea does not > dissolve and is in crystalline form only, will it have any effect on > solubilzation efficiency. Our solubilization time is 1 Hr and after that > we > centrifuge and use the supernatant for refolding via dialysis. however the > pellet after centrifugation of solubilzation show presence of our protein > on > sds page analysis. what should we do so that the process of solubilization > is complete and our protein is not lost in pellet. > > thanks in anticipation. > > meg > E-mail message checked by Spyware Doctor (6.1.0.447) Database version: 6.13830 http://www.pctools.com/en/spyware-doctor-antivirus/